Background MicroRNA (miRNA) and other small regulatory RNAs contribute to the modulation of a large number of cellular processes. reads originated from the loop regions of the precursors of two previously reported miRNAs (bmo-miR-1920 and miR-1921). Interestingly, the majority of the newly identified miRNAs were silkworm-specific, 23 unique miRNAs were widely conserved from invertebrates to vertebrates, 13 unique miRNAs were limited to invertebrates, and 32 were confined to insects. We identified 24 closely positioned clusters and 45 paralogs of miRNAs in the silkworm genome. However, sequence tags showed that paralogs or clusters were not prerequisites for coordinated transcription and accumulation. The majority of silkworm-specific miRNAs were located in transposable elements, and displayed significant differences in abundance between the anterior-middle and posterior silk gland. Conclusions Conservative analysis revealed that miRNAs can serve as phylogenetic markers and function in evolutionary signaling. The newly identified miRNAs greatly enrich the repertoire of insect miRNAs, and provide insights into miRNA SB 525334 evolution, biogenesis, and expression in insects. The differential expression of miRNAs in the anterior-middle and posterior silk glands supports their involvement as new levels in the regulation of the silkworm silk gland. Background Following their initial discovery in worms, an increasing number of 18-30 nt-sized small RNAs have been identified as crucial regulatory molecules in multicellular organisms, animal viruses, and unicellular organisms [1-7]. Identification of abundant miRNAs and other small regulatory RNAs in different organisms is critical in improving our understanding of genome organization, genome biology, and evolution [8]. The silkworm, Bombx mori SB 525334 (B. mori), an important model organism used to investigate several fundamental biological phenomena (including development, gene regulation, and morphological innovation [9]), has been employed for silk production for about 5,000 years. The recently sequenced B. mori is the first lepidopteran insect genome that provides a resource for Lum comparative genomics studies, facilitating our understanding of insect evolution [10]. The latest miRNA database release (miRBase 14.0) presents 91 silkworm miRNAs and two so-called miRNA* sequences originating from the RNA hairpin arm opposite the annotated mature miRNA-containing arm [2,11]. However, some of these miRNAs have been identified solely on the basis of sequence similarity to known orthologs, and have never been confirmed experimentally. Furthermore, the total number of silkworm miRNA genes is significantly lower than that in fruit fly (152) and human (701), and it is likely that further miRNAs remain to be discovered in the silkworm. To extend the known repertoire of small regulatory RNAs expressed in the silkworm, we constructed and sequenced three small RNA libraries prepared from the whole body (WB) as well as the anterior-middle and posterior silk glands (AMSG and PSG) of day-3 fifth instar larvae. The silk gland of B. mori is differentiated into anterior, middle, and posterior sections [12,13]. Expression of all sericin genes is limited to the anterior and middle parts of the middle silk gland [14,15], whereas the fibroin genes are expressed exclusively in the posterior silk gland [16,17]. Both sericin and fibroin genes are topologically and temporally regulated at the transcriptional level in a concerted manner during larval development [18,19]. The spatial distribution of miRNAs may contribute to the mechanistic understanding of concerted silk protein synthesis. Each library was individually sequenced, and generated more than 5 million short reads, resulting in a total of 36 million reads, of which 1,819,103 were miRNA reads. The newly identified miRNAs significantly enhance our knowledge of insect miRNA species and provide insights into miRNA evolution, biogenesis, and expression in insects. Results Overall complexity of small RNA pools between the libraries We obtained raw data by sequencing three small RNA pools of the whole SB 525334 silkworm body from 5th-instar day-3 larvae, and anterior-middle and posterior silkworm silk SB 525334 glands, using the latest sequencing Solexa technology [8,20], filtered the low quality reads according to base quality value, trimmed the adaptor sequence at the 3′ primer terminus, cleaned up 5′ adaptor contaminants formed by ligation, and finally collected the small RNAs and analyzed size distribution. The raw data and processed files of the three libraries have been deposited in NCBI’s Gene Expression Omnibus (GEO) [21,22] under accession number GSE 17965. For analysis, all identical sequence reads in each small RNA library were grouped and converted into unique sequences with associated counts of the individual reads. The flow results of data filtration for the three libraries are presented in Additional file 1. The total number of raw sequence reads in the whole body small RNA library is 5,467,768, comprising 2,848,263 low-quality reads (52.09%) and 2,619,505 high-quality reads (47.91%)..